The proposed studies will investigate the molecular basis of acute virulence in Toxoplasma gondii taking advantage of forward and reverse genetics to identify genes important for pathogenesis in the murine model. Our previous studies have shown that T. gondii has a highly unusual genetic makeup consisting of three widespread, predominant clonal lineages. Type I strains are acutely virulent in mice and are more commonly associated with some forms of human toxoplasmosis. Genetic linkage mapping was used to identify several quantitative trait loci (QTLs) that control acute virulence in the type I lineage. The most prominent of these is a locus on chromosome VII that accounts for about 50% of acute virulence. A partially overlapping locus controls the ability of the parasite to cross biological barriers and disseminate in vivo. The central hypothesis for our studies is that genes found in the type I lineage control acute virulence, which is mediated in part by active migration across biological barriers. In the proposed studies, we will identify genes that are important for acute virulence and determine the cellular and molecular basis for their effects. First, we will generate a high resolution linkage map of T. gondii and anchor these markers to the available genome sequence. Second, we will employ this high-resolution map to more precisely locate QTLs associated with acute virulence (VIR) and enhanced migration (MIG). Finally, we will test the role of these candidate virulence genes using molecular genetics to generate transgenic and knockout parasite lines. These studies should provide important fundamental knowledge about the molecular basis of parasite virulence that is relevant to human toxoplasmosis in the immunocompromised patient. [unreadable] [unreadable] [unreadable]